PROJECT SUMMARY This application is submitted in response to the NCI Provocative Questions (PQ) Initiative to address PQ #3, ?How do variations in tumor-associated immune responses contribute to differences in cancer risk, incidence, or progression?? The intent of this PQ is to encourage research to study the causes of variable tumor- associated immune responses and/or how these variations relate to cancer risk, incidence, or progression. Our focus is osteosarcoma, a rare disease that primarily impacts children and adolescents. The research area is important to address significant unmet health needs for osteosarcoma patients with regard to the etiology of the disease, strategies to manage progression, and methods to guide personalized treatments. We chose to use the exploratory R21 mechanism as a means for hypothesis-testing and hypothesis generation from a massive sequencing effort through which we have generated a unique resource of primary and metastatic samples from humans, dogs, and mice with osteosarcoma. The effort includes sequencing of germ line DNA to identify de novo and acquired mutations, transcriptomes and methylomes. To complement these primary and autochthonous samples, we have developed robust models to study the osteosarcoma progression using xenografts as well as the role of the immune system in fully immunocompetent mice. We have assembled a robust team with diverse and comprehensive expertise in sarcoma biology and therapy, pediatric oncology, epidemiology and statistics, veterinary medicine and pathology, animal models, genetics and genomic engineering, inflammation, immunology and immunotherapy, molecular biology, transcription, microRNA networks, and systems biology and bioinformatics. The proposal includes intellectual and technical innovations in the multi-species comparative approach integrating multiple human and animal models, as well as in the conceptual frameworks that will be tested. Specifically we will investigate two issues that are directly pertinent to the PQ: For specific aim 1, we will investigate if there are correlations between inflammatory and immune cell infiltrates in the tumor environment and the biological behavior of the tumors. Data from this aim will inform the case for innate tumor traits as significant drivers of the immune response, allowing us to postulate a causality hypothesis for test it in a future larger project. Specific aim 2 is designed to determine the mechanisms that drive patterning of immune infiltrates in osteosarcoma. Data from this aim, whether conserved or species-specific, will confirm a new and exciting mechanism of tumor-mediated immunomodulation that could be used as a biomarker, and that also could be therapeutically tractable. Overall, the project has the potential to impact patient management and selection for clinical trials evaluating sarcoma immunotherapy.